Methods of Improving Accuracy and Precision of Droplet Metering Using an On-Actuator Reservoir as the Fluid Input

ABSTRACT

The present invention is directed to methods of improving accuracy of droplet metering using at least one on-actuator reservoir as the fluid input. In some embodiments, the on-actuator reservoir that is used for metering droplets includes a loading port, a liquid storage zone, a droplet metering zone, and a droplet dispensing zone. The on-actuator reservoirs are designed to prevent liquid flow-back into the loading port and to prevent liquid from flooding into the droplet operations gap in the dispensing zone.

1. FIELD OF THE INVENTION

The invention relates to methods and systems for improving accuracy ofdroplet metering using an on-actuator reservoir as the fluid input.

2. BACKGROUND

A droplet actuator typically includes one or more substrates configuredto form a surface or gap for conducting droplet operations. The one ormore substrates establish a droplet operations surface or gap forconducting droplet operations and may also include electrodes arrangedto conduct the droplet operations. The droplet operations substrate orthe gap between the substrates may be coated or filled with a fillerfluid that is immiscible with the liquid that forms the droplets. It canbe difficult to control the size and volume of droplets dispensed in adroplet actuator. There is a need for new approaches to accuratelydispensing droplets in a droplet actuator.

3. BRIEF DESCRIPTION OF THE INVENTION

A droplet actuator is provided, including at least one on-actuatorreservoir, the at least one on-actuator reservoir including: a) aloading port; b) a liquid storage zone; c) a droplet metering zone; andd) a droplet dispensing zone; where the at least one on-actuatorreservoir is designed for improving the accuracy of droplet metering. Incertain embodiments, the droplet dispensing zone includes a topsubstrate and a bottom substrate separated to form a droplet operationsgap. In other embodiments, the bottom substrate includes a dropletprocessing region, particularly where the droplet processing regionincludes at least one arrangement of droplet operations electrodesdisposed on the bottom substrate. In further embodiments, the at leastone arrangement of droplet operations electrodes includes at least onearrangement of electrowetting-mediated droplet operations electrodes. Inother embodiments, a plurality of sets of reservoir electrodes aredisposed on the bottom substrate. In still further embodiments, anon-actuator reservoir is formed in the top substrate at each set ofreservoir electrodes. In other embodiments, the metering zone is a bulkliquid metering zone.

In another embodiment, the storage zone, metering zone, and dispensingzone of the on-actuator reservoir of the droplet actuator are eachcharacterized by different gap heights. In certain embodiments, the gapheight of the storage zone is h1, the gap height of the metering zone ish2, and the gap height of the dispensing zone is h3. In otherembodiments, h1>h2>h3. In other embodiments, h1 is about 3 mm, h2 isabout 800 μm, and h3 is about 300 μm, particularly where the capacity ofthe on-actuator reservoir is about 61.58 μL or about 47.45 μL. Infurther embodiments, h1 is about 2400 μm, h2 is about 800 μm, and h3 isabout 300 μm, particularly where the capacity of the on-actuatorreservoir is about 45.17 μL or about 33.25 μL. In other embodiments, h1is about 200 μm, h2 is about 800 μm, and h3 is about 300 μm,particularly where the capacity of the on-actuator reservoir is about35.37 μL or about 25.09 μL. In further embodiments, h1 is about 1600 μm,h2 is about 1000 μm, and h3 is about 312.5 μm, particularly where thecapacity of the on-actuator reservoir is about 26.48 μL. In otherembodiments, h1 is about 3000 μm, h2 is about 1600 μm, and h3 is about312.5 μm, particularly where the capacity of the on-actuator reservoiris about 61.58 μL. In further embodiments, h1 is about 3000 μm, h2 isabout 1200 μm, and h3 is about 312.5 μm, particularly where the capacityof the on-actuator reservoir is about 62.58 μL. In other embodiments, h1is about 2000 μm, h2 is about 1000 μm, and h3 is about 300 μm,particularly where the capacity of the on-actuator reservoir is about25.09 μL. In further embodiments, h1 is about 3000 μm, h2 is about 1600μm, and h3 is about 300 μm, particularly where the capacity of theon-actuator reservoir is about 47.45 μL. In other embodiments, h1 isabout 3000 μm, h2 is about 1200 μm, and h3 is about 300 μm, particularlywhere the capacity of the on-actuator reservoir is about 47.45 μL. Infurther embodiments, h1 is about 800 μm, h2 is about 800 μm, and h3 isabout 300 μm. In other embodiments, the length of the dispensing zone isselected from the group consisting of 1.5× the height h2, 2× the heighth2, 2.5× the height h2, and 3× the height h2. In further embodiments,the width of the dispensing zone is selected from the group consistingof 1.5× the height h2, 2× the height h2, 2.5× the height h2, and 3× theheight h2. In still further embodiments, the length and the width of thedispensing zone are each independently selected from the groupconsisting of 1.5× the height h2, 2× the height h2, 2.5× the height h2,and 3× the height h2.

In a further embodiment, the top substrate of the droplet actuatorincludes a transition region for transitioning the gap height from themetering zone to the dispensing zone. In another embodiment, thetransition region includes a slope in the surface of the top substratethat is facing the droplet operations gap, particularly where the slopeis about 45 degrees.

In another embodiment, the loading port of the droplet actuator includesa cup portion for holding a volume of liquid. In a further embodiment,the cup portion is fitted upon an upwardly protruding outlet portion. Inanother embodiment, the cup portion includes an upper portion, furtherwhere the upper portion is enclosed but includes an opening therein. Ina further embodiment, the opening of the upper portion of the cupportion is substantially triangular in footprint. In another embodiment,the outlet portion includes an opening therein. In a further embodiment,the opening of the outlet portion is substantially circular infootprint. In another embodiment, the cup portion of the loading port isfilled with liquid at least up to the height of the outlet portion ofthe loading port. In a further embodiment, a pressure loading source iscoupled to the opening of the cup portion of the loading port.

In another embodiment, the loading port of the droplet actuator includesan upper portion and a lower portion, where the upper portion is open.In a further embodiment, the lower portion of the loading port includesan outlet that allows liquid to flow into the droplet operations gap. Inanother embodiment, a pressure loading source is coupled to the outletof the loading port. In a further embodiment, the pressure loadingsource is a pipette including a pipette tip, particularly where theoutlet of the loading port is designed for the pipette tip to be fittedtherein.

In another embodiment, the droplet actuator further includes a pluralityof droplet processing lanes, where the droplet processing lanes areformed by and fluidly connected by the at least one arrangement ofdroplet operations electrodes, particularly where the plurality ofdroplet processing lanes includes eight droplet processing lanes.

In a further embodiment, the relationship of each set of reservoirelectrodes to the on-actuator reservoir formed in the top substrate ateach set of reservoir electrodes is such that the larger segments of thereservoir electrodes are oriented toward the storage zone of theon-actuator reservoir and the smaller segments of each set of reservoirelectrodes are oriented toward the dispensing zone of the on-actuatorreservoir.

In another embodiment, the diameter of the opening leading from theloading port into the storage zone in the droplet actuator is smallenough compared to the liquid storage area to prevent liquid flow-backinto the space above the storage zone. In a further embodiment, thedesign of the loading port and the storage zone prevents liquidflow-back onto the outside surface of the top substrate, particularlywhere the outside surface of the top substrate does not comprise a CYTOPcoating. In another embodiment, the at least one on-actuator reservoirprevents liquid from flooding into the droplet operations gap in thedispensing zone. In a further embodiment, the bottom substrate of thedroplet actuator comprises a set of power/signal I/O pads patterned atone end thereof.

In another embodiment, the at least one reservoir of the dropletactuator supplies the droplet processing region, particularly whereinthe at least one reservoir is a sample reservoir or a reagent reservoir.In a further embodiment, each set of reservoir electrodes supports anon-actuator reservoir, particularly where the plurality of sets ofreservoir electrodes comprises seven or eight sets of reservoirelectrodes. In another embodiment, the droplet processing regionsupplies at least one collection or waste reservoir. In a furtherembodiment, the droplet processing region supplies a plurality of setsof reservoir electrodes.

Methods of improving accuracy of droplet metering on a droplet actuatorare also provided using a droplet actuator as disclosed herein, wherethe droplet actuator comprises a loading port that includes a cupportion for holding a volume of liquid, where the cup portion is fittedupon an upwardly protruding outlet portion, and where the cup portionincludes an upper portion, further where the upper portion is enclosedbut includes an opening therein. The method comprises the steps of: a)Coupling the pressure loading source to the opening of the cup portionof the loading port; b) Flowing sufficient liquid into the storage zoneto fill the storage zone without causing liquid to flow into thedispensing zone or creating enough pressure to permit the liquid to flowinto the metering zone and/or the dispensing zone or creating enoughpressure to cause the liquid to escape back through the loading port tothe exterior of droplet actuator; c) Metering sub-droplets from liquidin the storage zone into the metering zone using the reservoirelectrodes in the metering zone to yield metered droplets in themetering zone; and d) Dispensing sub-droplets from the metered dropletsin the metering zone, using reservoir electrodes in the dispensing zoneto dispense sub-droplets onto the droplet operations electrodes; wherethe method provides for accurate droplet metering on a droplet actuator.In a particular embodiment, the cup portion of the loading port isfilled with liquid at least up to the height of the outlet portion ofthe loading port. In a further embodiment, a pressure loading source iscoupled to the opening of the cup portion of the loading port. In someembodiments, the metering zone is a bulk liquid metering zone,particularly where relatively constant pressure of a volume of bulkliquid in the bulk liquid metering zone is maintained prior to dropletdispensing. In other embodiments, metering of a bulk liquid in the bulkliquid metering zone prior to droplet dispensing comprises dispensing ofa single droplet or dispensing of multiple droplets. In a furtherembodiment, the at least one on-actuator reservoir is designed toprevent liquid flow-back into the loading port. In another embodiment,the diameter of the opening leading from the loading port into thestorage zone is small enough compared to the liquid storage area toprevent liquid flow-back into the space above the storage zone. In afurther embodiment, the design of the loading port and the storage zoneprevents liquid flow-back onto the outside surface of the top substrate.In another embodiment, the at least one on-actuator reservoir preventsliquid from flooding into the droplet operations gap in the dispensingzone. In a further embodiment, modular changes are made to the design ofthe functional zones to provide increased liquid processing capacityand/or to provide for different zone and/or gap heights without the needfor changing the entire droplet actuator and/or system design.

A method of improving accuracy of droplet metering on a droplet actuatoris also provided, where the droplet actuator comprises any of thedroplet actuators as disclosed herein, further where the loading portcomprises an upper portion and a lower portion, where the upper portionis open. The method comprises the steps of: a) Coupling the pressureloading source to the outlet of the loading port; b) Flowing sufficientliquid into the storage zone to fill the storage zone without causingliquid to flow into the dispensing zone or creating enough pressure topermit the liquid to flow into the metering zone and/or the dispensingzone or creating enough pressure to cause the liquid to escape backthrough the loading port to the exterior of droplet actuator; c)Metering sub-droplets from liquid in the storage zone into the meteringzone using the reservoir electrodes in the metering zone to yieldmetered droplets in the metering zone; and d) Dispensing sub-dropletsfrom the metered droplets in the metering zone, using reservoirelectrodes in the dispensing zone to dispense sub-droplets onto thedroplet operations electrodes; wherein the method provides for accuratedroplet metering on a droplet actuator. In one embodiment, the pressureloading source is a pipette comprising a pipette tip, further whereinthe outlet of the loading port is designed for the pipette tip to befitted therein. In another embodiment, the metering zone is a bulkliquid metering zone. In some embodiments, the metering zone is a bulkliquid metering zone, particularly where relatively constant pressure ofa volume of bulk liquid in the bulk liquid metering zone is maintainedprior to droplet dispensing. In other embodiments, metering of a bulkliquid in the bulk liquid metering zone prior to droplet dispensingcomprises dispensing of a single droplet or dispensing of multipledroplets. In a further embodiment, the at least one on-actuatorreservoir is designed to prevent liquid flow-back into the loading port.In another embodiment, the diameter of the opening leading from theloading port into the storage zone is small enough compared to theliquid storage area to prevent liquid flow-back into the space above thestorage zone. In a further embodiment, the design of the loading portand the storage zone prevents liquid flow-back onto the outside surfaceof the top substrate. In another embodiment, the at least oneon-actuator reservoir prevents liquid from flooding into the dropletoperations gap in the dispensing zone. In a further embodiment, modularchanges are made to the design of the functional zones to provideincreased liquid processing capacity and/or to provide for differentzone and/or gap heights without the need for changing the entire dropletactuator and/or system design.

A microfluidics system is also provided, where the microfluidics systemis programmed to execute any of the methods as described herein on anyof the droplet actuators as described herein.

A storage medium is also provided, where the storage medium comprisesprogram code embodied in the medium for executing any of the methods asdescribed herein on any of the droplet actuators as described herein.

A microfluidics system is also provided, where the microfluidics systemcomprises any of the droplet actuators as described herein coupled to aprocessor, particularly where the processor executes program codeembodied in a storage medium for executing any of the methods asdescribed herein.

These and other embodiments are described more fully below.

4. DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate,” with reference to one or more electrodes, means affecting achange in the electrical state of the one or more electrodes which, inthe presence of a droplet, results in a droplet operation. Activation ofan electrode can be accomplished using alternating or direct current.Any suitable voltage may be used. For example, an electrode may beactivated using a voltage which is greater than about 150 V, or greaterthan about 200 V, or greater than about 250 V, or from about 275 V toabout 1000 V, or about 300 V. Where alternating current is used, anysuitable frequency may be employed. For example, an electrode may beactivated using alternating current having a frequency from about 1 Hzto about 10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hzto about 40 Hz, or about 30 Hz.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical, amorphous and other three dimensional shapes. The bead may, forexample, be capable of being subjected to a droplet operation in adroplet on a droplet actuator or otherwise configured with respect to adroplet actuator in a manner which permits a droplet on the dropletactuator to be brought into contact with the bead on the dropletactuator and/or off the droplet actuator. Beads may be provided in adroplet, in a droplet operations gap, or on a droplet operationssurface. Beads may be provided in a reservoir that is external to adroplet operations gap or situated apart from a droplet operationssurface, and the reservoir may be associated with a flow path thatpermits a droplet including the beads to be brought into a dropletoperations gap or into contact with a droplet operations surface. Beadsmay be manufactured using a wide variety of materials, including forexample, resins, and polymers. The beads may be any suitable size,including for example, microbeads, microparticles, nanobeads andnanoparticles. In some cases, beads are magnetically responsive; inother cases beads are not significantly magnetically responsive. Formagnetically responsive beads, the magnetically responsive material mayconstitute substantially all of a bead, a portion of a bead, or only onecomponent of a bead. The remainder of the bead may include, among otherthings, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable beads include flowcytometry microbeads, polystyrene microparticles and nanoparticles,functionalized polystyrene microparticles and nanoparticles, coatedpolystyrene microparticles and nanoparticles, silica microbeads,fluorescent microspheres and nanospheres, functionalized fluorescentmicrospheres and nanospheres, coated fluorescent microspheres andnanospheres, color dyed microparticles and nanoparticles, magneticmicroparticles and nanoparticles, superparamagnetic microparticles andnanoparticles (e.g., DYNABEADS® particles, available from InvitrogenGroup, Carlsbad, Calif.), fluorescent microparticles and nanoparticles,coated magnetic microparticles and nanoparticles, ferromagneticmicroparticles and nanoparticles, coated ferromagnetic microparticlesand nanoparticles, and those described in U.S. Patent Publication Nos.20050260686, entitled “Multiplex flow assays preferably with magneticparticles as solid phase,” published on Nov. 24, 2005; 20030132538,entitled “Encapsulation of discrete quanta of fluorescent particles,”published on Jul. 17, 2003; 20050118574, entitled “Multiplexed Analysisof Clinical Specimens Apparatus and Method,” published on Jun. 2, 2005;20050277197. Entitled “Microparticles with Multiple Fluorescent Signalsand Methods of Using Same,” published on Dec. 15, 2005; 20060159962,entitled “Magnetic Microspheres for use in Fluorescence-basedApplications,” published on Jul. 20, 2006; the entire disclosures ofwhich are incorporated herein by reference for their teaching concerningbeads and magnetically responsive materials and beads. Beads may bepre-coupled with a biomolecule or other substance that is able to bindto and form a complex with a biomolecule. Beads may be pre-coupled withan antibody, protein or antigen, DNA/RNA probe or any other moleculewith an affinity for a desired target. Examples of droplet actuatortechniques for immobilizing magnetically responsive beads and/ornon-magnetically responsive beads and/or conducting droplet operationsprotocols using beads are described in U.S. patent application Ser. No.11/639,566, entitled “Droplet-Based Particle Sorting,” filed on Dec. 15,2006; U.S. Patent Application No. 61/039,183, entitled “MultiplexingBead Detection in a Single Droplet,” filed on Mar. 25, 2008; U.S. PatentApplication No. 61/047,789, entitled “Droplet Actuator Devices andDroplet Operations Using Beads,” filed on Apr. 25, 2008; U.S. PatentApplication No. 61/086,183, entitled “Droplet Actuator Devices andMethods for Manipulating Beads,” filed on Aug. 5, 2008; InternationalPatent Application No. PCT/US2008/053545, entitled “Droplet ActuatorDevices and Methods Employing Magnetic Beads,” filed on Feb. 11, 2008;International Patent Application No. PCT/US2008/058018, entitled“Bead-based Multiplexed Analytical Methods and Instrumentation,” filedon Mar. 24, 2008; International Patent Application No.PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on Mar.23, 2008; and International Patent Application No. PCT/US2006/047486,entitled “Droplet-based Biochemistry,” filed on Dec. 11, 2006; theentire disclosures of which are incorporated herein by reference. Beadcharacteristics may be employed in the multiplexing aspects of theinvention. Examples of beads having characteristics suitable formultiplexing, as well as methods of detecting and analyzing signalsemitted from such beads, may be found in U.S. Patent Publication No.20080305481, entitled “Systems and Methods for Multiplex Analysis of PCRin Real Time,” published on Dec. 11, 2008; U.S. Patent Publication No.20080151240, “Methods and Systems for Dynamic Range Expansion,”published on Jun. 26, 2008; U.S. Patent Publication No. 20070207513,entitled “Methods, Products, and Kits for Identifying an Analyte in aSample,” published on Sep. 6, 2007; U.S. Patent Publication No.20070064990, entitled “Methods and Systems for Image Data Processing,”published on Mar. 22, 2007; U.S. Patent Publication No. 20060159962,entitled “Magnetic Microspheres for use in Fluorescence-basedApplications,” published on Jul. 20, 2006; U.S. Patent Publication No.20050277197, entitled “Microparticles with Multiple Fluorescent Signalsand Methods of Using Same,” published on Dec. 15, 2005; and U.S. PatentPublication No. 20050118574, entitled “Multiplexed Analysis of ClinicalSpecimens Apparatus and Method,” published on Jun. 2, 2005.

“Droplet” means a volume of liquid on a droplet actuator. Typically, adroplet is at least partially bounded by a filler fluid. For example, adroplet may be completely surrounded by a filler fluid or may be boundedby filler fluid and one or more surfaces of the droplet actuator. Asanother example, a droplet may be bounded by filler fluid, one or moresurfaces of the droplet actuator, and/or the atmosphere. As yet anotherexample, a droplet may be bounded by filler fluid and the atmosphere.Droplets may, for example, be aqueous or non-aqueous or may be mixturesor emulsions including aqueous and non-aqueous components. Droplets maytake a wide variety of shapes; nonlimiting examples include generallydisc shaped, slug shaped, truncated sphere, ellipsoid, spherical,partially compressed sphere, hemispherical, ovoid, cylindrical,combinations of such shapes, and various shapes formed during dropletoperations, such as merging or splitting or formed as a result ofcontact of such shapes with one or more surfaces of a droplet actuator.For examples of droplet fluids that may be subjected to dropletoperations using the approach of the invention, see International PatentApplication No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,”filed on Dec. 11, 2006. In various embodiments, a droplet may include abiological sample, such as whole blood, lymphatic fluid, serum, plasma,sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid,seminal fluid, vaginal excretion, serous fluid, synovial fluid,pericardial fluid, peritoneal fluid, pleural fluid, transudates,exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid,fecal samples, liquids containing single or multiple cells, liquidscontaining organelles, fluidized tissues, fluidized organisms, liquidscontaining multi-celled organisms, biological swabs and biologicalwashes. Moreover, a droplet may include a reagent, such as water,deionized water, saline solutions, acidic solutions, basic solutions,detergent solutions and/or buffers. Other examples of droplet contentsinclude reagents, such as a reagent for a biochemical protocol, such asa nucleic acid amplification protocol, an affinity-based assay protocol,an enzymatic assay protocol, a sequencing protocol, and/or a protocolfor analyses of biological fluids. A droplet may include one or morebeads.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplet actuators, see Pamula et al., U.S. Pat. No.6,911,132, entitled “Apparatus for Manipulating Droplets byElectrowetting-Based Techniques,” issued on Jun. 28, 2005; Pamula etal., U.S. patent application Ser. No. 11/343,284, entitled “Apparatusesand Methods for Manipulating Droplets on a Printed Circuit Board,” filedon filed on Jan. 30, 2006; Pollack et al., International PatentApplication No. PCT/US2006/047486, entitled “Droplet-BasedBiochemistry,” filed on Dec. 11, 2006; Shenderov, U.S. Pat. No.6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No.6,565,727, entitled “Actuators for Microfluidics Without Moving Parts,”issued on Jan. 24, 2000; Kim and/or Shah et al., U.S. patent applicationSer. No. 10/343,261, entitled “Electrowetting-driven Micropumping,”filed on Jan. 27, 2003, Ser. No. 11/275,668, entitled “Method andApparatus for Promoting the Complete Transfer of Liquid Drops from aNozzle,” filed on Jan. 23, 2006, Ser. No. 11/460,188, entitled “SmallObject Moving on Printed Circuit Board,” filed on Jan. 23, 2006, Ser.No. 12/465,935, entitled “Method for Using Magnetic Particles in DropletMicrofluidics,” filed on May 14, 2009, and Ser. No. 12/513,157, entitled“Method and Apparatus for Real-time Feedback Control of ElectricalManipulation of Droplets on Chip,” filed on Apr. 30, 2009; Velev, U.S.Pat. No. 7,547,380, entitled “Droplet Transportation Devices and MethodsHaving a Fluid Surface,” issued on Jun. 16, 2009; Sterling et al., U.S.Pat. No. 7,163,612, entitled “Method, Apparatus and Article forMicrofluidic Control via Electrowetting, for Chemical, Biochemical andBiological Assays and the Like,” issued on Jan. 16, 2007; Becker andGascoyne et al., U.S. Pat. No. 7,641,779, entitled “Method and Apparatusfor Programmable fluidic Processing,” issued on Jan. 5, 2010, and U.S.Pat. No. 6,977,033, entitled “Method and Apparatus for Programmablefluidic Processing,” issued on Dec. 20, 2005; Decre et al., U.S. Pat.No. 7,328,979, entitled “System for Manipulation of a Body of Fluid,”issued on Feb. 12, 2008; Yamakawa et al., U.S. Patent Pub. No.20060039823, entitled “Chemical Analysis Apparatus,” published on Feb.23, 2006; Wu, International Patent Pub. No. WO/2009/003184, entitled“Digital Microfluidics Based Apparatus for Heat-exchanging ChemicalProcesses,” published on Dec. 31, 2008; Fouillet et al., U.S. PatentPub. No. 20090192044, entitled “Electrode Addressing Method,” publishedon Jul. 30, 2009; Fouillet et al., U.S. Pat. No. 7,052,244, entitled“Device for Displacement of Small Liquid Volumes Along a Micro-catenaryLine by Electrostatic Forces,” issued on May 30, 2006; Marchand et al.,U.S. Patent Pub. No. 20080124252, entitled “Droplet Microreactor,”published on May 29, 2008; Adachi et al., U.S. Patent Pub. No.20090321262, entitled “Liquid Transfer Device,” published on Dec. 31,2009; Roux et al., U.S. Patent Pub. No. 20050179746, entitled “Devicefor Controlling the Displacement of a Drop Between two or Several SolidSubstrates,” published on Aug. 18, 2005; Dhindsa et al., “VirtualElectrowetting Channels: Electronic Liquid Transport with ContinuousChannel Functionality,” Lab Chip, 10:832-836 (2010); the entiredisclosures of which are incorporated herein by reference, along withtheir priority documents. Certain droplet actuators will include one ormore substrates arranged with a droplet operations gap therebetween andelectrodes associated with (e.g., layered on, attached to, and/orembedded in) the one or more substrates and arranged to conduct one ormore droplet operations. For example, certain droplet actuators willinclude a base (or bottom) substrate, droplet operations electrodesassociated with the substrate, one or more dielectric layers atop thesubstrate and/or electrodes, and optionally one or more hydrophobiclayers atop the substrate, dielectric layers and/or the electrodesforming a droplet operations surface. A top substrate may also beprovided, which is separated from the droplet operations surface by agap, commonly referred to as a droplet operations gap. Various electrodearrangements on the top and/or bottom substrates are discussed in theabove-referenced patents and applications and certain novel electrodearrangements are discussed in the description of the invention. Duringdroplet operations it is preferred that droplets remain in continuouscontact or frequent contact with a ground or reference electrode. Aground or reference electrode may be associated with the top substratefacing the gap, the bottom substrate facing the gap, in the gap. Whereelectrodes are provided on both substrates, electrical contacts forcoupling the electrodes to a droplet actuator instrument for controllingor monitoring the electrodes may be associated with one or both plates.In some cases, electrodes on one substrate are electrically coupled tothe other substrate so that only one substrate is in contact with thedroplet actuator. In one embodiment, a conductive material (e.g., anepoxy, such as MASTER BOND™ Polymer System EP79, available from MasterBond, Inc., Hackensack, N.J.) provides the electrical connection betweenelectrodes on one substrate and electrical paths on the othersubstrates, e.g., a ground electrode on a top substrate may be coupledto an electrical path on a bottom substrate by such a conductivematerial. Where multiple substrates are used, a spacer may be providedbetween the substrates to determine the height of the gap therebetweenand define dispensing reservoirs. The spacer height may, for example, befrom about 5 μm to about 600 μm, or about 100 μm to about 400 μm, orabout 200 μm to about 350 μm, or about 250 μm to about 300 μm, or about275 μm. The spacer may, for example, be formed of a layer of projectionsform the top or bottom substrates, and/or a material inserted betweenthe top and bottom substrates. One or more openings may be provided inthe one or more substrates for forming a fluid path through which liquidmay be delivered into the droplet operations gap. The one or moreopenings may in some cases be aligned for interaction with one or moreelectrodes, e.g., aligned such that liquid flowed through the openingwill come into sufficient proximity with one or more droplet operationselectrodes to permit a droplet operation to be effected by the dropletoperations electrodes using the liquid. The base (or bottom) and topsubstrates may in some cases be formed as one integral component. One ormore reference electrodes may be provided on the base (or bottom) and/ortop substrates and/or in the gap. Examples of reference electrodearrangements are provided in the above referenced patents and patentapplications. In various embodiments, the manipulation of droplets by adroplet actuator may be electrode mediated, e.g., electrowettingmediated or dielectrophoresis mediated or Coulombic force mediated.Examples of other techniques for controlling droplet operations that maybe used in the droplet actuators of the invention include using devicesthat induce hydrodynamic fluidic pressure, such as those that operate onthe basis of mechanical principles (e.g. external syringe pumps,pneumatic membrane pumps, vibrating membrane pumps, vacuum devices,centrifugal forces, piezoelectric/ultrasonic pumps and acoustic forces);electrical or magnetic principles (e.g. electroosmotic flow,electrokinetic pumps, ferrofluidic plugs, electrohydrodynamic pumps,attraction or repulsion using magnetic forces and magnetohydrodynamicpumps); thermodynamic principles (e.g. gas bubblegeneration/phase-change-induced volume expansion); other kinds ofsurface-wetting principles (e.g. electrowetting, and optoelectrowetting,as well as chemically, thermally, structurally and radioactively inducedsurface-tension gradients); gravity; surface tension (e.g., capillaryaction); electrostatic forces (e.g., electroosmotic flow); centrifugalflow (substrate disposed on a compact disc and rotated); magnetic forces(e.g., oscillating ions causes flow); magnetohydrodynamic forces; andvacuum or pressure differential. In certain embodiments, combinations oftwo or more of the foregoing techniques may be employed to conduct adroplet operation in a droplet actuator of the invention. Similarly, oneor more of the foregoing may be used to deliver liquid into a dropletoperations gap, e.g., from a reservoir in another device or from anexternal reservoir of the droplet actuator (e.g., a reservoir associatedwith a droplet actuator substrate and a flow path from the reservoirinto the droplet operations gap). Droplet operations surfaces of certaindroplet actuators of the invention may be made from hydrophobicmaterials or may be coated or treated to make them hydrophobic. Forexample, in some cases some portion or all of the droplet operationssurfaces may be derivatized with low surface-energy materials orchemistries, e.g., by deposition or using in situ synthesis usingcompounds such as poly- or per-fluorinated compounds in solution orpolymerizable monomers. Examples include TEFLON® AF (available fromDuPont, Wilmington, Del.), members of the cytop family of materials,coatings in the FLUOROPEL® family of hydrophobic and superhydrophobiccoatings (available from Cytonix Corporation, Beltsville, Md.), silanecoatings, fluorosilane coatings, hydrophobic phosphonate derivatives(e.g., those sold by Aculon, Inc), and NOVEC™ electronic coatings(available from 3M Company, St. Paul, Minn.), other fluorinated monomersfor plasma-enhanced chemical vapor deposition (PECVD), andorganosiloxane (e.g., SiOC) for PECVD. In some cases, the dropletoperations surface may include a hydrophobic coating having a thicknessranging from about 10 nm to about 1,000 nm. Moreover, in someembodiments, the top substrate of the droplet actuator includes anelectrically conducting organic polymer, which is then coated with ahydrophobic coating or otherwise treated to make the droplet operationssurface hydrophobic. For example, the electrically conducting organicpolymer that is deposited onto a plastic substrate may bepoly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS).Other examples of electrically conducting organic polymers andalternative conductive layers are described in Pollack et al.,International Patent Application No. PCT/US2010/040705, entitled“Droplet Actuator Devices and Methods,” the entire disclosure of whichis incorporated herein by reference. One or both substrates may befabricated using a printed circuit board (PCB), glass, indium tin oxide(ITO)-coated glass, and/or semiconductor materials as the substrate.When the substrate is ITO-coated glass, the ITO coating is preferably athickness in the range of about 20 to about 200 nm, preferably about 50to about 150 nm, or about 75 to about 125 nm, or about 100 nm. In somecases, the top and/or bottom substrate includes a PCB substrate that iscoated with a dielectric, such as a polyimide dielectric, which may insome cases also be coated or otherwise treated to make the dropletoperations surface hydrophobic. When the substrate includes a PCB, thefollowing materials are examples of suitable materials: MITSUI™ BN-300(available from MITSUI Chemicals America, Inc., San Jose Calif.); ARLON™11N (available from Arlon, Inc, Santa Ana, Calif.).; NELCO® N4000-6 andN5000-30/32 (available from Park Electrochemical Corp., Melville, N.Y.);ISOLA™ FR406 (available from Isola Group, Chandler, Ariz.), especiallyIS620; fluoropolymer family (suitable for fluorescence detection sinceit has low background fluorescence); polyimide family; polyester;polyethylene naphthalate; polycarbonate; polyetheretherketone; liquidcrystal polymer; cyclo-olefin copolymer (COC); cyclo-olefin polymer(COP); aramid; THERMOUNT® nonwoven aramid reinforcement (available fromDuPont, Wilmington, Del.); NOMEX® brand fiber (available from DuPont,Wilmington, Del.); and paper. Various materials are also suitable foruse as the dielectric component of the substrate. Examples include:vapor deposited dielectric, such as PARYLENE™ C (especially on glass),PARYLENE™ N, and PARYLENE™ HT (for high temperature, ˜300° C.)(available from Parylene Coating Services, Inc., Katy, Tex.); TEFLON® AFcoatings; cytop; soldermasks, such as liquid photoimageable soldermasks(e.g., on PCB) like TAIYO™ PSR4000 series, TAIYO™ PSR and AUS series(available from Taiyo America, Inc. Carson City, Nev.) (good thermalcharacteristics for applications involving thermal control), andPROBIMER™ 8165 (good thermal characteristics for applications involvingthermal control (available from Huntsman Advanced Materials AmericasInc., Los Angeles, Calif.); dry film soldermask, such as those in theVACREL® dry film soldermask line (available from DuPont, Wilmington,Del.); film dielectrics, such as polyimide film (e.g., KAPTON® polyimidefilm, available from DuPont, Wilmington, Del.), polyethylene, andfluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester;polyethylene naphthalate; cyclo-olefin copolymer (COC); cyclo-olefinpolymer (COP); any other PCB substrate material listed above; blackmatrix resin; and polypropylene. Droplet transport voltage and frequencymay be selected for performance with reagents used in specific assayprotocols. Design parameters may be varied, e.g., number and placementof on-actuator reservoirs, number of independent electrode connections,size (volume) of different reservoirs, placement of magnets/bead washingzones, electrode size, inter-electrode pitch, and gap height (betweentop and bottom substrates) may be varied for use with specific reagents,protocols, droplet volumes, etc. In some cases, a substrate of theinvention may derivatized with low surface-energy materials orchemistries, e.g., using deposition or in situ synthesis using poly- orper-fluorinated compounds in solution or polymerizable monomers.Examples include TEFLON® AF coatings and FLUOROPEL® coatings for dip orspray coating, other fluorinated monomers for plasma-enhanced chemicalvapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD.Additionally, in some cases, some portion or all of the dropletoperations surface may be coated with a substance for reducingbackground noise, such as background fluorescence from a PCB substrate.For example, the noise-reducing coating may include a black matrixresin, such as the black matrix resins available from Toray industries,Inc., Japan. Electrodes of a droplet actuator are typically controlledby a controller or a processor, which is itself provided as part of asystem, which may include processing functions as well as data andsoftware storage and input and output capabilities. Reagents may beprovided on the droplet actuator in the droplet operations gap or in areservoir fluidly coupled to the droplet operations gap. The reagentsmay be in liquid form, e.g., droplets, or they may be provided in areconstitutable form in the droplet operations gap or in a reservoirfluidly coupled to the droplet operations gap. Reconstitutable reagentsmay typically be combined with liquids for reconstitution. An example ofreconstitutable reagents suitable for use with the invention includesthose described in Meathrel, et al., U.S. Pat. No. 7,727,466, entitled“Disintegratable films for diagnostic devices,” granted on Jun. 1, 2010.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations that are sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to volume of the resulting droplets(i.e., the volume of the resulting droplets can be the same ordifferent) or number of resulting droplets (the number of resultingdroplets may be 2, 3, 4, 5 or more). The term “mixing” refers to dropletoperations which result in more homogenous distribution of one or morecomponents within a droplet. Examples of “loading” droplet operationsinclude microdialysis loading, pressure assisted loading, roboticloading, passive loading, and pipette loading. Droplet operations may beelectrode-mediated. In some cases, droplet operations are furtherfacilitated by the use of hydrophilic and/or hydrophobic regions onsurfaces and/or by physical obstacles. For examples of dropletoperations, see the patents and patent applications cited above underthe definition of “droplet actuator.” Impedance or capacitance sensingor imaging techniques may sometimes be used to determine or confirm theoutcome of a droplet operation. Examples of such techniques aredescribed in Sturmer et al., International Patent Pub. No.WO/2008/101194, entitled “Capacitance Detection in a Droplet Actuator,”published on Aug. 21, 2008, the entire disclosure of which isincorporated herein by reference. Generally speaking, the sensing orimaging techniques may be used to confirm the presence or absence of adroplet at a specific electrode. For example, the presence of adispensed droplet at the destination electrode following a dropletdispensing operation confirms that the droplet dispensing operation waseffective. Similarly, the presence of a droplet at a detection spot atan appropriate step in an assay protocol may confirm that a previous setof droplet operations has successfully produced a droplet for detection.Droplet transport time can be quite fast. For example, in variousembodiments, transport of a droplet from one electrode to the next mayexceed about 1 sec, or about 0.1 sec, or about 0.01 sec, or about 0.001sec. In one embodiment, the electrode is operated in AC mode but isswitched to DC mode for imaging. It is helpful for conducting dropletoperations for the footprint area of droplet to be similar toelectrowetting area; in other words, 1×-, 2×-3×-droplets are usefullycontrolled operated using 1, 2, and 3 electrodes, respectively. If thedroplet footprint is greater than the number of electrodes available forconducting a droplet operation at a given time, the difference betweenthe droplet size and the number of electrodes should typically not begreater than 1; in other words, a 2× droplet is usefully controlledusing 1 electrode and a 3× droplet is usefully controlled using 2electrodes. When droplets include beads, it is useful for droplet sizeto be equal to the number of electrodes controlling the droplet, e.g.,transporting the droplet.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. For example, the dropletoperations gap of a droplet actuator is typically filled with a fillerfluid. The filler fluid may, for example, be or include a low-viscosityoil, such as silicone oil or hexadecane filler fluid. The filler fluidmay be or include a halogenated oil, such as a fluorinated orperfluorinated oil. The filler fluid may fill the entire gap of thedroplet actuator or may coat one or more surfaces of the dropletactuator. Filler fluids may be conductive or non-conductive. Fillerfluids may be selected to improve droplet operations and/or reduce lossof reagent or target substances from droplets, improve formation ofmicrodroplets, reduce cross contamination between droplets, reducecontamination of droplet actuator surfaces, reduce degradation ofdroplet actuator materials, etc. For example, filler fluids may beselected for compatibility with droplet actuator materials. As anexample, fluorinated filler fluids may be usefully employed withfluorinated surface coatings. Fluorinated filler fluids are useful toreduce loss of lipophilic compounds, such as umbelliferone substrateslike 6-hexadecanoylamido-4-methylumbelliferone substrates (e.g., for usein Krabbe, Niemann-Pick, or other assays); other umbelliferonesubstrates are described in U.S. Patent Pub. No. 20110118132, publishedon May 19, 2011, the entire disclosure of which is incorporated hereinby reference. Examples of suitable fluorinated oils include those in theGalden line, such as Galden HT170 (bp=170° C., viscosity=1.8 cSt,density=1.77), Galden HT200 (bp=200 C, viscosity=2.4 cSt, d=1.79),Galden HT230 (bp=230 C, viscosity=4.4 cSt, d=1.82) (all from SolvaySolexis); those in the Novec line, such as Novec 7500 (bp=128 C,viscosity=0.8 cSt, d=1.61), Fluorinert FC-40 (bp=155° C., viscosity=1.8cSt, d=1.85), Fluorinert FC-43 (bp=174° C., viscosity=2.5 cSt, d=1.86)(both from 3M). In general, selection of perfluorinated filler fluids isbased on kinematic viscosity (<7 cSt is preferred, but not required),and on boiling point (>150° C. is preferred, but not required, for usein DNA/RNA-based applications (PCR, etc.)). Filler fluids may, forexample, be doped with surfactants or other additives. For example,additives may be selected to improve droplet operations and/or reduceloss of reagent or target substances from droplets, formation ofmicrodroplets, cross contamination between droplets, contamination ofdroplet actuator surfaces, degradation of droplet actuator materials,etc. Composition of the filler fluid, including surfactant doping, maybe selected for performance with reagents used in the specific assayprotocols and effective interaction or non-interaction with dropletactuator materials. Examples of filler fluids and filler fluidformulations suitable for use with the invention are provided inSrinivasan et al, International Patent Pub. Nos. WO/2010/027894,entitled “Droplet Actuators, Modified Fluids and Methods,” published onMar. 11, 2010, and WO/2009/021173, entitled “Use of Additives forEnhancing Droplet Operations,” published on Feb. 12, 2009; Sista et al.,International Patent Pub. No. WO/2008/098236, entitled “Droplet ActuatorDevices and Methods Employing Magnetic Beads,” published on Aug. 14,2008; and Monroe et al., U.S. Patent Publication No. 20080283414,entitled “Electrowetting Devices,” filed on May 17, 2007; the entiredisclosures of which are incorporated herein by reference, as well asthe other patents and patent applications cited herein. Fluorinated oilsmay in some cases be doped with fluorinated surfactants, e.g., ZonylFSO-100 (Sigma-Aldrich) and/or others.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position in a dropletto permit execution of a droplet splitting operation, yielding onedroplet with substantially all of the beads and one dropletsubstantially lacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, and CoMnP.

“Reservoir” means an enclosure or partial enclosure configured forholding, storing, or supplying liquid. A droplet actuator system of theinvention may include on-cartridge reservoirs and/or off-cartridgereservoirs. On-cartridge reservoirs may be (1) on-actuator reservoirs,which are reservoirs in the droplet operations gap or on the dropletoperations surface; (2) off-actuator reservoirs, which are reservoirs onthe droplet actuator cartridge, but outside the droplet operations gap,and not in contact with the droplet operations surface; or (3) hybridreservoirs which have on-actuator regions and off-actuator regions. Anexample of an off-actuator reservoir is a reservoir in the topsubstrate. An off-actuator reservoir is typically in fluid communicationwith an opening or flow path arranged for flowing liquid from theoff-actuator reservoir into the droplet operations gap, such as into anon-actuator reservoir. An off-cartridge reservoir may be a reservoirthat is not part of the droplet actuator cartridge at all, but whichflows liquid to some portion of the droplet actuator cartridge. Forexample, an off-cartridge reservoir may be part of a system or dockingstation to which the droplet actuator cartridge is coupled duringoperation. Similarly, an off-cartridge reservoir may be a reagentstorage container or syringe which is used to force fluid into anon-cartridge reservoir or into a droplet operations gap. A system usingan off-cartridge reservoir will typically include a fluid passage meanswhereby liquid may be transferred from the off-cartridge reservoir intoan on-cartridge reservoir or into a droplet operations gap.

“Transporting into the magnetic field of a magnet,” “transportingtowards a magnet,” and the like, as used herein to refer to dropletsand/or magnetically responsive beads within droplets, is intended torefer to transporting into a region of a magnetic field capable ofsubstantially attracting magnetically responsive beads in the droplet.Similarly, “transporting away from a magnet or magnetic field,”“transporting out of the magnetic field of a magnet,” and the like, asused herein to refer to droplets and/or magnetically responsive beadswithin droplets, is intended to refer to transporting away from a regionof a magnetic field capable of substantially attracting magneticallyresponsive beads in the droplet, whether or not the droplet ormagnetically responsive beads is completely removed from the magneticfield. It will be appreciated that in any of such cases describedherein, the droplet may be transported towards or away from the desiredregion of the magnetic field, and/or the desired region of the magneticfield may be moved towards or away from the droplet. Reference to anelectrode, a droplet, or magnetically responsive beads being “within” or“in” a magnetic field, or the like, is intended to describe a situationin which the electrode is situated in a manner which permits theelectrode to transport a droplet into and/or away from a desired regionof a magnetic field, or the droplet or magnetically responsive beadsis/are situated in a desired region of the magnetic field, in each casewhere the magnetic field in the desired region is capable ofsubstantially attracting any magnetically responsive beads in thedroplet. Similarly, reference to an electrode, a droplet, ormagnetically responsive beads being “outside of” or “away from” amagnetic field, and the like, is intended to describe a situation inwhich the electrode is situated in a manner which permits the electrodeto transport a droplet away from a certain region of a magnetic field,or the droplet or magnetically responsive beads is/are situated awayfrom a certain region of the magnetic field, in each case where themagnetic field in such region is not capable of substantially attractingany magnetically responsive beads in the droplet or in which anyremaining attraction does not eliminate the effectiveness of dropletoperations conducted in the region. In various aspects of the invention,a system, a droplet actuator, or another component of a system mayinclude a magnet, such as one or more permanent magnets (e.g., a singlecylindrical or bar magnet or an array of such magnets, such as a Halbacharray) or an electromagnet or array of electromagnets, to form amagnetic field for interacting with magnetically responsive beads orother components on chip. Such interactions may, for example, includesubstantially immobilizing or restraining movement or flow ofmagnetically responsive beads during storage or in a droplet during adroplet operation or pulling magnetically responsive beads out of adroplet.

“Washing” with respect to washing a bead means reducing the amountand/or concentration of one or more substances in contact with the beador exposed to the bead from a droplet in contact with the bead. Thereduction in the amount and/or concentration of the substance may bepartial, substantially complete, or even complete. The substance may beany of a wide variety of substances; examples include target substancesfor further analysis, and unwanted substances, such as components of asample, contaminants, and/or excess reagent. In some embodiments, awashing operation begins with a starting droplet in contact with amagnetically responsive bead, where the droplet includes an initialamount and initial concentration of a substance. The washing operationmay proceed using a variety of droplet operations. The washing operationmay yield a droplet including the magnetically responsive bead, wherethe droplet has a total amount and/or concentration of the substancewhich is less than the initial amount and/or concentration of thesubstance. Examples of suitable washing techniques are described inPamula et al., U.S. Pat. No. 7,439,014, entitled “Droplet-Based SurfaceModification and Washing,” granted on Oct. 21, 2008, the entiredisclosure of which is incorporated herein by reference.

The terms “top,” “bottom,” “over,” “under,” and “on” are used throughoutthe description with reference to the relative positions of componentsof the droplet actuator, such as relative positions of top and bottomsubstrates of the droplet actuator. It will be appreciated that thedroplet actuator is functional regardless of its orientation in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface. In one example, fillerfluid can be considered as a film between such liquid and theelectrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a top down view of a bottom substrate of a dropletactuator that includes electrode arrangements that support on-actuatorreservoirs for accurately metering droplets;

FIGS. 2A and 2B illustrate a top down view and a cross-sectional view,respectively, of a portion of a droplet actuator, which shows an exampleof an on-actuator reservoir for accurately metering droplets;

FIGS. 3A and 3B illustrate a top down view and a cross-sectional view,respectively, of another portion of the droplet actuator, which showsanother example of an on-actuator reservoir for accurately meteringdroplets;

FIG. 4 illustrates a side view of another example of yet another portionof the droplet actuator, which shows yet another example of anon-actuator reservoir for accurately metering droplets; and

FIG. 5 illustrates a functional block diagram of an example of amicrofluidics system that includes a droplet actuator.

6. DESCRIPTION

The present invention is directed to methods of improving accuracy andprecision of droplet metering using an on-actuator reservoir as thefluid input. In some embodiments, the on-actuator reservoir that is usedfor metering droplets includes a loading port, a liquid storage zone, adroplet metering zone, and a droplet dispensing zone. The on-actuatorreservoir is designed to prevent liquid flow-back into the loading portand to prevent liquid from flooding into the droplet operations gap inthe dispensing zone.

6.1 on-Actuator Reservoirs

FIG. 1 illustrates a top down view of a bottom substrate 100 of adroplet actuator (not shown) that includes electrode arrangements thatsupport on-actuator reservoirs for accurately metering droplets. Forexample, bottom substrate 100 includes a set of power/signal I/O pads110 patterned at one end thereof, as shown. An electrode arrangement 120is also patterned on bottom substrate 100. Electrode arrangement 120includes a droplet processing region 122 that includes, for example, aplurality of droplet processing lanes that are formed by and fluidlyconnected by various arrangements of droplet operations electrodes 124(e.g., electrowetting electrodes). In one embodiment, the plurality ofdroplet processing lanes comprise eight droplet processing lanes.Additionally, various reservoirs (e.g., sample and reagent reservoirs)may supply droplet processing region 122. For example, electrodearrangement 120 includes a plurality of sets of reservoir electrodes126, wherein each set of reservoir electrodes 126 supports anon-actuator reservoir, an example of which is shown and described withreference to FIGS. 2A and 2B. In one embodiment, the plurality of setsof reservoir electrodes 126 comprise seven sets of reservoir electrodes126. Electrode arrangement 120 also includes a plurality of sets ofreservoir electrodes 128, wherein each set of reservoir electrodes 128supports an on-actuator reservoir, an example of which is shown anddescribed with reference to FIGS. 3A and 3B. Further, droplet processingregion 122 supplies certain collection or waste reservoirs. Accordingly,droplet processing region 122 supplies a plurality of sets of reservoirelectrodes 130, wherein each set of reservoir electrodes 130 supports anon-actuator reservoir (not shown). In one embodiment, the plurality ofsets of reservoir electrodes 130 comprise eight sets of reservoirelectrodes 130.

In electrode arrangement 120, each set of reservoir electrodes 126 andreservoir electrodes 128 supports an on-actuator reservoir that isdesigned for improving the accuracy and precision of droplet metering(i.e., droplet dispensing) into, for example, the droplet processingregion 122 of a droplet actuator (not shown). More details ofembodiments of on-actuator reservoirs that are designed for improvingthe accuracy and precision of droplet metering are described below withreference to FIGS. 2A, 2B, 3A, 3B, and 4.

FIGS. 2A and 2B illustrate a top down view and a cross-sectional view,respectively, of a portion of a droplet actuator 200, which shows anembodiment of an on-actuator reservoir for accurately metering droplets.Namely, FIG. 2B is a cross-sectional view taken along line A-A of FIG.2A. Droplet actuator 200 may include the bottom substrate 100 of FIG. 1along with a top substrate 212. Bottom substrate 100 and top substrate212 are separated by a droplet operations gap 214. Droplet operationsare conducted in the droplet operations gap 214 on a droplet operationssurface. Droplet actuator 200 includes the electrode arrangement 120disposed on bottom substrate 100, whereas the electrode arrangement 120includes the droplet operations electrodes 124. A ground referenceelectrode (not shown) may be disposed on top substrate 212. Dropletoperations electrodes 124 and the ground reference electrode arearranged for conducting droplet operations.

The portion of droplet actuator 200 shown in FIGS. 2A and 2B correspondsto one of the sets of reservoir electrodes 126 of bottom substrate 100shown in FIG. 1. Namely, an on-actuator reservoir 220 is formed in topsubstrate 212 at each set of reservoir electrodes 126. The on-actuatorreservoir 220 includes a loading port 222, a storage zone 224, ametering zone 226, and a dispensing zone 228. In one embodiment, themetering zone 226 is a bulk liquid metering zone. The storage zone 224,metering zone 226, and dispensing zone 228 are characterized bydifferent gap heights (i.e., different heights of the droplet operationsgap 214 in these zones). For example, storage zone 224 has a height h1,metering zone 226 has a height h2, and dispensing zone 228 has a heighth3, wherein h1>h2>h3. In one embodiment, height h1 is about 3 mm, heighth2 is about 800 μm, and height h3 is about 300 μm.

In one embodiment, there is a transition region 229 in top substrate 212for transitioning the gap height from metering zone 226 to dispensingzone 228. In transition region 229, there may be a slope in the surfaceof the top substrate 212 that is facing the droplet operations gap 214.In one embodiment, this slope is about 45 degrees.

Further, loading port 222 includes a cup portion 230 for holding avolume of liquid, wherein the cup portion 230 is fitted upon an upwardlyprotruding outlet portion 232. The upper portion of the cup portion 230is enclosed, but has an opening 234 therein. In one embodiment, opening234 of cup portion 230 of loading port 222 is substantially triangularin footprint. The outlet portion 232 has an opening 236 therein. In oneembodiment, opening 236 of outlet portion 232 of loading port 222 issubstantially circular in footprint. When in use, the cup portion 230 ofloading port 222 must be filled with liquid at least up to the height ofthe outlet portion 232 of loading port 222 in order for liquid to flowthrough opening 236 and into the droplet operations gap 214. A pressureloading source can be coupled to opening 234 of cup portion 230 ofloading port 222. Liquid can fill the cup portion 230 at least up to theheight of the outlet portion 232 of loading port 222 in order for liquidto flow through opening 236 and into the droplet operations gap 214.

The relationship of the reservoir electrodes 126 to on-actuatorreservoir 220 is such that the larger segments of the reservoirelectrodes 126 are oriented toward the storage zone 224 of on-actuatorreservoir 220 and the smaller segments of the reservoir electrodes 126are oriented toward the dispensing zone 228 of on-actuator reservoir220, which feeds the droplet operations electrodes 124, as shown.

FIGS. 3A and 3B illustrate a top down view and a cross-sectional view,respectively, of another portion of droplet actuator 200, which showsanother embodiment of an on-actuator reservoir for accurately meteringdroplets. Namely, FIG. 3B is a cross-sectional view taken along line A-Aof FIG. 3A.

The portion of droplet actuator 200 shown in FIGS. 3A and 3B correspondsto one of the sets of reservoir electrodes 128 of bottom substrate 100shown in FIG. 1. Namely, an on-actuator reservoir 320 is formed in topsubstrate 212 at each set of reservoir electrodes 128. The on-actuatorreservoir 320 includes a loading port 322, a storage zone 324, ametering zone 326, and a dispensing zone 328. In one embodiment, themetering zone 326 is a bulk liquid metering zone. The storage zone 324,metering zone 326, and dispensing zone 328 are characterized bydifferent gap heights (i.e., different heights of the droplet operationsgap 214 in these zones). For example, storage zone 324 has a height h1,metering zone 326 has a height h2, and dispensing zone 328 has a heighth3, wherein h1>h2>h3. In one embodiment, height h1 is about 3 mm, heighth2 is about 800 μm, and height h3 is about 300 μm.

Further, loading port 322 includes a cup portion 330 for holding avolume of liquid, wherein the cup portion 330 is fitted upon an upwardlyprotruding outlet portion 332. The upper portion of the cup portion 330is enclosed, but has an opening 334 therein. In one embodiment, opening334 of cup portion 330 of loading port 322 is substantially triangularin footprint. The outlet portion 332 has an opening 336 therein. In oneembodiment, opening 336 of outlet portion 332 of loading port 322 issubstantially circular in footprint. When in use, the cup portion 330 ofloading port 322 must be filled with liquid at least up to the height ofthe outlet portion 332 of loading port 322 in order for liquid to flowthrough opening 336 and into the droplet operations gap 214. A pressureloading source can be coupled to opening 334 of cup portion 330 ofloading port 322. Liquid can fill the cup portion 330 at least up to theheight of the outlet portion 332 of loading port 322 in order for liquidto flow through opening 336 and into the droplet operations gap 214.

The relationship of the reservoir electrodes 128 to on-actuatorreservoir 320 is such that the larger segments of the reservoirelectrodes 128 are oriented toward the storage zone 324 of on-actuatorreservoir 320 and the smaller segments of the reservoir electrodes 128are oriented toward the dispensing zone 328 of on-actuator reservoir320, which feeds the droplet operations electrodes 124, as shown.

The on-actuator reservoir 220 of FIGS. 2A and 2B and on-actuatorreservoir 320 of FIGS. 3A and 3B are sized differently as shown by thedifferent sized layouts of reservoir electrodes 126 and reservoirelectrodes 128 in FIG. 1. In this embodiment, the lengths of the storagezone 224, metering zone 226, and dispensing zone 228 of on-actuatorreservoir 220 is different than the lengths of the storage zone 324,metering zone 326, and dispensing zone 328, respectively, of on-actuatorreservoir 320. In one embodiment, the metering zone 326 is a bulk liquidmetering zone. An embodiment of the specifications of on-actuatorreservoir 220 of FIGS. 2A and 2B are shown below in Table 1. Anembodiment of the specifications of on-actuator reservoir 320 of FIGS.3A and 3B are shown below in Table 2.

TABLE 1 Example specifications of on-actuator reservoir 220 Height h1Height h2 Height h3 Estimated (μm) of (μm) of (μm) of capacity (μL) ofstorage metering dispensing on-actuator zone 224 zone 226 zone 228reservoir 220 3000 800 300 61.58 2400 800 300 45.17 2000 800 300 35.371600 1000 312.5 26.48 3000 1600 312.5 61.58 3000 1200 312.5 62.58 800800 300

TABLE 2 Example specifications of on-actuator reservoir 320 Height h1Height h2 Height h3 Estimated (μm) of (μm) of (μm) of capacity (μL) ofstorage metering dispensing on-actuator zone 324 zone 326 zone 328reservoir 320 3000 800 300 47.45 2400 800 300 33.25 2000 800 300 25.092000 1000 300 25.09 3000 1600 300 47.45 3000 1200 300 47.45 800 800 300

FIG. 4 illustrates a side view of another embodiment of yet anotherportion of droplet actuator 200, which shows yet another embodiment ofan on-actuator reservoir for accurately metering droplets. Namely, anon-actuator reservoir 420 is formed in top substrate 212. Theon-actuator reservoir 420 includes a loading port 422, a storage zone424, a metering zone 426, and a dispensing zone 428. In one embodiment,the metering zone 426 is a bulk liquid metering zone. The storage zone424, metering zone 426, and dispensing zone 428 are characterized bydifferent gap heights (i.e., different heights of the droplet operationsgap 214 in these zones). For example, storage zone 424 has a height h1,metering zone 426 has a height h2, and dispensing zone 428 has a heighth3, wherein h1>h2>h3.

Further, the upper portion of loading port 422 may be open and the lowerportion of loading port 422 may have an outlet 430 that allows liquid toflow into the droplet operations gap 214. In one embodiment, outlet 430of loading port 422 is designed for the tip of a pipette to be fittedtightly therein. In this way, the pipette tip can be used for pressureloading liquid into the droplet operations gap 214.

Referring now to FIGS. 1 through 4, there is a relationship between thelength, width, and height of each of the dispensing zones (e.g.,dispensing zones 226, 326, 426). For example, preferably the length ofthe dispensing zones is 1.5× the height h2, 2× the height h2, 2.5× theheight h2, or 3× the height h2. Further, preferably the width of thedispensing zones is 1.5× the height h2, 2× the height h2, 2.5× theheight h2, or 3× the height h2.

Additionally, the diameter of the openings leading from the loadingports (e.g., loading ports 222, 322, 422) into the storage zones (e.g.,storage zones 224, 324, 424) are small enough compared to the liquidstorage area to prevent liquid flow-back into the space above thestorage zones. Further, the design of the loading ports (e.g., loadingports 222, 322, 422) and storage zones (e.g., storage zones 224, 324,424) prevents liquid flow-back onto the outside surface of the topsubstrate (e.g., top substrate 212). Without liquid flow-back onto thetop substrate, the CYTOP coating (not shown) on the outside surface ofthe top substrate can be eliminated.

Referring now again to FIGS. 1 through 4, an embodiment of a method ofusing the presently disclosed on-actuator reservoirs for accuratelymetering droplets in a droplet actuator includes, but is not limited to,the following steps.

-   -   1. Coupling a pressure loading source to the loading fitting. In        one embodiment, in FIGS. 2A and 2B, a pressure loading source is        coupled to opening 234 of cup portion 230 of loading port 222.        In another embodiment, in FIG. 4, coupling, a pressure loading        source is coupled directly to outlet 430 of loading port 422.    -   2. Flowing into the storage zone sufficient liquid to fill the        zone without causing liquid to flow into the dispensing zone or        creating enough pressure to permit the liquid to escape through        the fitting opening to the exterior of the droplet actuator. For        example and referring now to FIGS. 2A and 2B, a sufficient        amount of liquid is flowed into storage zone 224 to fill storage        zone 224 without causing the liquid to flow into metering zone        226 and/or dispensing zone 228 or creating enough pressure to        cause the liquid to escape back through the loading port 222 to        the exterior of droplet actuator 200. In one embodiment, the        metering zone 226 is a bulk liquid metering zone.    -   3. Metering sub-droplets from the stored liquid using electrodes        in the metering zone to yield metered droplets. For example and        referring now to FIGS. 2A and 2B, from the bulk liquid in        storage zone 224, sub-droplets are metered into metering zone        226 using the reservoir electrodes 126 that are in metering zone        226 to yield metered droplets.    -   4. Dispensing sub-droplets from the metered droplets using        electrodes in the dispensing zone. For example and referring now        to FIGS. 2A and 2B, using reservoir electrodes 126 in dispensing        zone 228, sub-droplets are dispensed onto the droplet operations        electrodes 124 from the metered droplets that are in metering        zone 226.

The present invention provides improved metering of droplets bymaintaining relatively constant pressure of a larger volume of bulkliquid prior to droplet dispensing. In one embodiment, a single meteringof a bulk liquid prior to droplet dispensing can comprise dispensing ofa single droplet or can comprise dispensing of multiple dropletsdepending on the volume of the premetered bulk liquid. By limiting thesizes of input ports for liquid loading (i.e., the loading ports) andthe storage zones, liquid flow-back onto the outside surface of the topsubstrate is prevented and the need for a hydrophobic coating on the onthe outside surface of the top substrate is eliminated. Furthermore, theseparation of functional zones in a single liquid reservoir can enablethe implementation of modular changes in design (e.g., for increasedliquid processing capacity and/or for different zone and gap heights)without the need for changing the entire droplet actuator and/or systemdesign.

6.2 Systems

FIG. 5 illustrates a functional block diagram of an embodiment of amicrofluidics system 530 that includes a droplet actuator 505. Digitalmicrofluidic technology conducts droplet operations on discrete dropletsin a droplet actuator, such as droplet actuator 505, by electricalcontrol of their surface tension (electrowetting). The droplets may besandwiched between two substrates of droplet actuator 505, a bottomsubstrate and a top substrate separated by a droplet operations gap. Thebottom substrate may include an arrangement of electrically addressableelectrodes. The top substrate may include a reference electrode planemade, for example, from conductive ink or indium tin oxide (ITO). Thebottom substrate and the top substrate may be coated with a hydrophobicmaterial. Alternatively, by limiting the sizes of input ports for liquidloading (i.e., the loading ports) and the storage zones, liquidflow-back onto the outside surface of the top substrate is prevented andthe need for a hydrophobic coating on the on the outside surface of thetop substrate is eliminated. Droplet operations are conducted in thedroplet operations gap. The space around the droplets (i.e., the gapbetween bottom and top substrates) may be filled with an immiscibleinert fluid, such as silicone oil, to prevent evaporation of thedroplets and to facilitate their transport within the device. Otherdroplet operations may be effected by varying the patterns of voltageactivation; examples include merging, splitting, mixing, and dispensingof droplets.

Droplet actuator 505 may be designed to fit onto an instrument deck (notshown) of microfluidics system 530. The instrument deck may hold dropletactuator 505 and house other droplet actuator features, such as, but notlimited to, one or more magnets and one or more heating devices. Forexample, the instrument deck may house one or more magnets 510, whichmay be permanent magnets. Optionally, the instrument deck may house oneor more electromagnets 515. Magnets 510 and/or electromagnets 515 arepositioned in relation to droplet actuator 505 for immobilization ofmagnetically responsive beads. Optionally, the positions of magnets 510and/or electromagnets 515 may be controlled by a motor 520.Additionally, the instrument deck may house one or more heating devices525 for controlling the temperature within, for example, certainreaction and/or washing zones of droplet actuator 505. In oneembodiment, heating devices 525 may be heater bars that are positionedin relation to droplet actuator 505 for providing thermal controlthereof.

A controller 530 of microfluidics system 530 is electrically coupled tovarious hardware components of the invention, such as droplet actuator505, electromagnets 515, motor 520, and heating devices 525, as well asto a detector 535, an impedance sensing system 540, and any other inputand/or output devices (not shown). Controller 530 controls the overalloperation of microfluidics system 530. Controller 530 may, for example,be a general purpose computer, special purpose computer, personalcomputer, or other programmable data processing apparatus. Controller530 serves to provide processing capabilities, such as storing,interpreting, and/or executing software instructions, as well ascontrolling the overall operation of the system. Controller 530 may beconfigured and programmed to control data and/or power aspects of thesedevices. For example, in one aspect, with respect to droplet actuator505, controller 530 controls droplet manipulation byactivating/deactivating electrodes.

Detector 535 may be an imaging system that is positioned in relation todroplet actuator 505. In one embodiment, the imaging system may includeone or more light-emitting diodes (LEDs) (i.e., an illumination source)and a digital image capture device, such as a charge-coupled device(CCD) camera.

Impedance sensing system 540 may be any circuitry for detectingimpedance at a specific electrode of droplet actuator 505. In oneembodiment, impedance sensing system 540 may be an impedancespectrometer. Impedance sensing system 540 may be used to monitor thecapacitive loading of any electrode, such as any droplet operationselectrode, with or without a droplet thereon. For examples of suitablecapacitance detection techniques, see Sturmer et al., U.S. PatentApplication Publication No. US20100194408, entitled “CapacitanceDetection in a Droplet Actuator,” published on Aug. 5, 2010; and Bournet al., U.S. Patent Publication No. US20030080143, entitled “System andMethod for Dispensing Liquids,” published on May 1, 2003; the entiredisclosures of which are incorporated herein by reference.

Droplet actuator 505 may include disruption device 545. Disruptiondevice 545 may include any device that promotes disruption (lysis) ofmaterials, such as tissues, cells and spores in a droplet actuator.Disruption device 545 may, for example, be a sonication mechanism, aheating mechanism, a mechanical shearing mechanism, a bead beatingmechanism, physical features incorporated into the droplet actuator 505,an electric field generating mechanism, a thermal cycling mechanism, andany combinations thereof. Disruption device 545 may be controlled bycontroller 530.

It will be appreciated that various aspects of the invention may beembodied as a method, system, computer readable medium, and/or computerprogram product. Aspects of the invention may take the form of hardwareembodiments, software embodiments (including firmware, residentsoftware, micro-code, etc.), or embodiments combining software andhardware aspects that may all generally be referred to herein as a“circuit,” “module,” or “system.” Furthermore, the methods of theinvention may take the form of a computer program product on acomputer-usable storage medium having computer-usable program codeembodied in the medium.

Any suitable computer useable medium may be utilized for softwareaspects of the invention. The computer-usable or computer-readablemedium may be, for example but not limited to, an electronic, magnetic,optical, electromagnetic, infrared, or semiconductor system, apparatus,device, or propagation medium. The computer readable medium may includetransitory and/or non-transitory embodiments. More specific embodiments(a non-exhaustive list) of the computer-readable medium would includesome or all of the following: an electrical connection having one ormore wires, a portable computer diskette, a hard disk, a random accessmemory (RAM), a read-only memory (ROM), an erasable programmableread-only memory (EPROM or Flash memory), an optical fiber, a portablecompact disc read-only memory (CD-ROM), an optical storage device, atransmission medium such as those supporting the Internet or anintranet, or a magnetic storage device. Note that the computer-usable orcomputer-readable medium could even be paper or another suitable mediumupon which the program is printed, as the program can be electronicallycaptured, via, for instance, optical scanning of the paper or othermedium, then compiled, interpreted, or otherwise processed in a suitablemanner, if necessary, and then stored in a computer memory. In thecontext of this document, a computer-usable or computer-readable mediummay be any medium that can contain, store, communicate, propagate, ortransport the program for use by or in connection with the instructionexecution system, apparatus, or device.

Program code for carrying out operations of the invention may be writtenin an object oriented programming language such as Java, Smalltalk, C++or the like. However, the program code for carrying out operations ofthe invention may also be written in conventional procedural programminglanguages, such as the “C” programming language or similar programminglanguages. The program code may be executed by a processor, applicationspecific integrated circuit (ASIC), or other component that executes theprogram code. The program code may be simply referred to as a softwareapplication that is stored in memory (such as the computer readablemedium discussed above). The program code may cause the processor (orany processor-controlled device) to produce a graphical user interface(“GUI”). The graphical user interface may be visually produced on adisplay device, yet the graphical user interface may also have audiblefeatures. The program code, however, may operate in anyprocessor-controlled device, such as a computer, server, personaldigital assistant, phone, television, or any processor-controlled deviceutilizing the processor and/or a digital signal processor.

The program code may locally and/or remotely execute. The program code,for example, may be entirely or partially stored in local memory of theprocessor-controlled device. The program code, however, may also be atleast partially remotely stored, accessed, and downloaded to theprocessor-controlled device. A user's computer, for example, mayentirely execute the program code or only partly execute the programcode. The program code may be a stand-alone software package that is atleast partly on the user's computer and/or partly executed on a remotecomputer or entirely on a remote computer or server. In the latterscenario, the remote computer may be connected to the user's computerthrough a communications network.

The invention may be applied regardless of networking environment. Thecommunications network may be a cable network operating in theradio-frequency domain and/or the Internet Protocol (IP) domain. Thecommunications network, however, may also include a distributedcomputing network, such as the Internet (sometimes alternatively knownas the “World Wide Web”), an intranet, a local-area network (LAN),and/or a wide-area network (WAN). The communications network may includecoaxial cables, copper wires, fiber optic lines, and/or hybrid-coaxiallines. The communications network may even include wireless portionsutilizing any portion of the electromagnetic spectrum and any signalingstandard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA or anycellular standard, and/or the ISM band). The communications network mayeven include powerline portions, in which signals are communicated viaelectrical wiring. The invention may be applied to any wireless/wirelinecommunications network, regardless of physical componentry, physicalconfiguration, or communications standard(s).

Certain aspects of invention are described with reference to variousmethods and method steps. It will be understood that each method stepcan be implemented by the program code and/or by machine instructions.The program code and/or the machine instructions may create means forimplementing the functions/acts specified in the methods.

The program code may also be stored in a computer-readable memory thatcan direct the processor, computer, or other programmable dataprocessing apparatus to function in a particular manner, such that theprogram code stored in the computer-readable memory produce or transforman article of manufacture including instruction means which implementvarious aspects of the method steps.

The program code may also be loaded onto a computer or otherprogrammable data processing apparatus to cause a series of operationalsteps to be performed to produce a processor/computer implementedprocess such that the program code provides steps for implementingvarious functions/acts specified in the methods of the invention.

7. CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. The term “theinvention” or the like is used with reference to certain specificexamples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation.

1. A droplet actuator, comprising: a. a top substrate and a bottomsubstrate separated to form a droplet operations gap therebetween; b. atleast one set of reservoir electrodes disposed on the bottom substrate;and c. at least one on-actuator reservoir formed in the top substrateand corresponding to the at least one set of reservoir electrodes, theat least one on-actuator reservoir comprising: i. a loading port; ii. aliquid storage zone; iii. a droplet metering zone; and iv. a dropletdispensing zone.
 2. (canceled)
 3. The droplet actuator of claim 1,wherein the bottom substrate comprises a droplet processing region. 4.The droplet actuator of claim 3, wherein the droplet processing regioncomprises at least one arrangement of droplet operations electrodesdisposed on the bottom substrate.
 5. The droplet actuator of claim 4,wherein the top substrate comprises a ground reference electrode.
 6. Thedroplet actuator of claim 4, wherein the at least one arrangement ofdroplet operations electrodes comprises at least one arrangement ofelectrowetting-mediated droplet operations electrodes.
 7. The dropletactuator of claim 1, wherein a plurality of sets of reservoir electrodesare disposed on the bottom substrate.
 8. (canceled)
 9. The dropletactuator of claim 1, wherein the metering zone is a bulk liquid meteringzone.
 10. The droplet actuator of claim 1, wherein the storage zone,metering zone, and dispensing zone are each characterized by differentgap heights.
 11. The droplet actuator of claim 10, wherein the gapheight of the storage zone is h1, the gap height of the metering zone ish2, and the gap height of the dispensing zone is h3.
 12. The dropletactuator of claim 11, wherein h1>h2>h3.
 13. The droplet actuator ofclaim 12, wherein h1 is about 3 mm, h2 is about 800 μm, and h3 is about300 μm.
 14. The droplet actuator of claim 13, wherein the capacity ofthe on-actuator reservoir is about 61.58 μL.
 15. (canceled)
 16. Thedroplet actuator of claim 12, wherein h1 is about 2400 μm, h2 is about800 μm, and h3 is about 300 μm.
 17. The droplet actuator of claim 16,wherein the capacity of the on-actuator reservoir is about 45.17 μL. 18.(canceled)
 19. The droplet actuator of claim 12, wherein h1 is about2000 μm, h2 is about 800 μm, and h3 is about 300 μm.
 20. The dropletactuator of claim 19, wherein the capacity of the on-actuator reservoiris about 35.37 μL.
 21. (canceled)
 22. The droplet actuator of claim 12,wherein h1 is about 1600 μm, h2 is about 1000 μm, and h3 is about 312.5μm.
 23. The droplet actuator of claim 22, wherein the capacity of theon-actuator reservoir is about 26.48 μL.
 24. The droplet actuator ofclaim 12, wherein h1 is about 3000 μm, h2 is about 1600 μm, and h3 isabout 312.5 μm.
 25. The droplet actuator of claim 24, wherein thecapacity of the on-actuator reservoir is about 61.58 μL.
 26. The dropletactuator of claim 12, wherein h1 is about 3000 μm, h2 is about 1200 μm,and h3 is about 312.5 μm.
 27. The droplet actuator of claim 26, whereinthe capacity of the on-actuator reservoir is about 62.58 μL. 28-33.(canceled)
 34. The droplet actuator of claim 11, wherein h1 is about 800μm, h2 is about 800 μm, and h3 is about 300 μm.
 35. The droplet actuatorof claim 11, wherein the length of the dispensing zone is selected fromthe group consisting of 1.5× the height h2, 2× the height h2, 2.5× theheight h2, and 3× the height h2.
 36. The droplet actuator of claim 11,wherein the width of the dispensing zone is selected from the groupconsisting of 1.5× the height h2, 2× the height h2, 2.5× the height h2,and 3× the height h2.
 37. The droplet actuator claim 10, wherein the topsubstrate comprises a transition region for transitioning the gap heightfrom the metering zone to the dispensing zone.
 38. The droplet actuatorof claim 37, wherein the transition region comprises a slope in thesurface of the top substrate that is facing the droplet operations gap.39. The droplet actuator of claim 38, wherein the slope is about 45degrees. 40-41. (canceled)
 42. The droplet actuator of claim 1, whereinthe relationship of the at least one set of reservoir electrodes to theat least one on-actuator reservoir formed in the top substrate at the atleast one set of reservoir electrodes is such that larger segments ofthe reservoir electrodes are oriented toward the storage zone of theon-actuator reservoir and smaller segments of each set of reservoirelectrodes are oriented toward the dispensing zone of the on-actuatorreservoir.
 43. The droplet actuator of claim 1, wherein a diameter of anopening leading from the loading port into the storage zone is smallenough compared to the liquid storage area to prevent liquid flow-backinto a space above the storage zone.
 44. The droplet actuator of claim1, wherein the design of the loading port and the storage zone preventsliquid flow-back onto an outside surface of the top substrate. 45.(canceled)
 46. The droplet actuator of claim 1, wherein the at least oneon-actuator reservoir prevents liquid from flooding into the dropletoperations gap in the dispensing zone. 47-50. (canceled)
 51. The dropletactuator of claim 1, wherein each of the at least one set of reservoirelectrodes supports an on-actuator reservoir. 52-55. (canceled)
 56. Thedroplet actuator of claim 1, wherein the loading port comprises a cupportion for holding a volume of liquid.
 57. The droplet actuator ofclaim 56, wherein the cup portion is fitted upon an upwardly protrudingoutlet portion.
 58. The droplet actuator of claim 56, wherein the cupportion comprises an upper portion, further wherein the upper portion isenclosed but comprises an opening therein.
 59. The droplet actuator ofclaim 58, wherein the opening of the upper portion of the cup portion issubstantially triangular in footprint.
 60. The droplet actuator of claim57, wherein the outlet portion comprises an opening therein.
 61. Thedroplet actuator of claim 60, wherein the opening of the outlet portionis substantially circular in footprint.
 62. The droplet actuator ofclaim 57, wherein the cup portion of the loading port is filled withliquid at least up to the height of the outlet portion of the loadingport.
 63. The droplet actuator of claim 56, wherein a pressure loadingsource is coupled to the opening of the cup portion of the loading port.64. The droplet actuator of claim 1, wherein the loading port comprisesan upper portion and a lower portion, wherein the upper portion is open.65. The droplet actuator of claim 64, wherein the lower portion of theloading port comprises an outlet that allows liquid to flow into thedroplet operations gap. 66-94. (canceled)